Despite the relatively ready availability of thyroid cyst fluid specimens, little has been published on their biochemical composition. We measured the concentrations of 18 analytes in thyroid cyst fluid specimens from benign (n=17) and malignant (n=3) lesions and in homogenates of normal thyroid tissue (n=5). The concentrations of an additional five analytes were measured in selected cyst fluid specimens only. Compared with normal human serum specimens, we found that in thyroid cyst fluid specimens the activities of acid phosphatase, aspartate aminotransferase, amylase, and lactate dehydrogenase, and the concentrations of iron and total bibilirubin were highly increased. The concentration of glucose was low. The gross appearance of the fluids and the presence of certain analytes were consistent with a hemorrhagic origin of most of the benign and malignant cyst fluid specimens. Other biochemical markers, however, indicated colloidlike features and/or an admixture of thyroid tissue components to the cyst fluid. Although we have limited data for cyst fluid specimens from malignant thyroid lesion, we found no evidence that the results of any of the common biochemical tests would distinguish benign from malignant lesions.